Measuring clathrin-mediated and independent endocytosis

Clathrin-mediated endocytosis was evaluated by measuring the uptake of transferrin from Human Serum, Alexa Fluor 488 Conjugate (T13342, Thermofisher Scientific). Clathrin-independent endocytosis was measured by intake of mouse monoclonal antibodies directed toward MHC-I (major histocompatibility complex I; clone w6/32, Biolegend) or control mouse monoclonal anti-cytokeratin 14 antibody (ab7800; Abcam; antibodies directed against intracellular target), followed by removal of the unbound antibody by low pH acid wash, fixation, and staining with secondary fluorescein isothiocyanate–labeled goat anti-mouse antibody (115-095; Jackson Laboratories).^{16 (link)} In some experiments, control (Adcontrol) and CLIC4-overexpressing (AdCLIC4) cells were treated with 20 mg/L protein synthesis inhibitor cycloheximide (CAS 66-81-9; Santa Cruz Biotechnology), with or without the endocytosis inhibitor Pitsop2 (20 μmol/L; ab120687; Abcam)^{16 (link)} or Pitstop2-negative control (20 μmol/L; ab120688; Abcam,) for 2 hours. Pitsop2-negative control is chemically related to Pitstop2 but does not block receptor-mediated endocytosis. BMPRII expression was analyzed by Western blotting.

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Abdul-Salam V.B., Russomanno G., Chien-Nien C., Mahomed A.S., Yates L.A., Wilkins M.R., Zhao L., Gierula M., Dubois O., Schaeper U., Endruschat J, & Wojciak-Stothard B. (2018). CLIC4/Arf6 Pathway: A New Lead in BMPRII Inhibition in Pulmonary Hypertension. Circulation Research, 124(1), 52-65.

Publication 2018

Alexa Fluor 488 Conjugate clone w6/32 ab7800 cycloheximide ab120687 ab120688

Acid Alexa fluor 488 Anti antibody Antibodies Antibody Block Bmprii Cells Clathrin Clic4 Clone Complex i Cycloheximide Cytokeratin 14 Endocytosis Fluorescein Goat Human Intracellular Isothiocyanate Monoclonal antibodies Mouse Protein synthesis inhibitor Serum Transferrin

Corresponding Organization :

Other organizations : Imperial College London, Silence Therapeutics (Germany)

Top 5 similar protocols

Variable analysis

independent variables

Overexpression of CLIC4 (AdCLIC4)
Treatment with protein synthesis inhibitor cycloheximide (CAS 66-81-9; 20 mg/L)
Treatment with endocytosis inhibitor Pitsop2 (20 μmol/L; ab120687; Abcam)
Treatment with Pitstop2-negative control (20 μmol/L; ab120688; Abcam)

dependent variables

Clathrin-mediated endocytosis (measured by uptake of transferrin from Human Serum, Alexa Fluor 488 Conjugate)
Clathrin-independent endocytosis (measured by intake of mouse monoclonal antibodies directed toward MHC-I or control mouse monoclonal anti-cytokeratin 14 antibody)
BMPRII expression (analyzed by Western blotting)

control variables

Control (Adcontrol) cells

positive controls

Mouse monoclonal antibodies directed toward MHC-I (major histocompatibility complex I; clone w6/32, Biolegend)

negative controls

Control mouse monoclonal anti-cytokeratin 14 antibody (ab7800; Abcam)

Annotations

Based on most similar protocols

The protocols use transferrin conjugated to fluorescent dyes (e.g. Texas Red, Alexa Fluor 488, Alexa Fluor 633) to measure clathrin-mediated endocytosis by quantifying the uptake of fluorescent transferrin (Protocols 1, 2, 3, 4).

For clathrin-independent endocytosis, the protocols use monoclonal antibodies directed against MHC-I or control antibodies, followed by removal of unbound antibody and staining with a secondary fluorescent antibody (Input protocol).

The protocols use pharmacological inhibitors to modulate endocytosis, such as chlorpromazine and Pitstop2 to inhibit clathrin-mediated endocytosis (Protocols 1, 3, Input protocol).

The protocols use various cell types, including Huh7.5 cells (Protocol 1), primary murine lymphatic endothelial cells (Protocol 2), and monocyte-derived macrophages (MDMs) (Protocol 3).

The protocols quantify endocytosis by measuring the fluorescence intensity of internalized transferrin or antibodies using techniques such as fluorescence microscopy (Protocols 2, 4), flow cytometry (Protocols 3, 4), or image analysis software (Protocol 1).

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